Insulin action on glucose uptake in fat and skeletal muscle tissues is mediated by translocation of glucose transporter Glut4 from an "intracellular storage pool" to the plasma membrane. The intracellular pool of Glut4 is not homogenous and consists of several distinct membrane compartments only one of which, insulin-responsive vesicles, or IRVs, is believed to be translocated to the plasma membrane upon insulin stimulation. The rest of intracellular Glut4 is localized in early and recycling endosomes as well as in intermediate transport vesicles. In the previous funding period, we developed a procedure for the biochemical separation of Glut4-containing endosomes from post-endosomal membrane vesicles. In addition, we identified a specific marker for one type of Glut4-containing vesicles, cellugyrin, and prepared antibodies to this protein. Thus, we can now separate individual intracellular Glut4-containing compartments and, for the first time, isolate pure IRVs. Based on these findings, we propose to determine their protein composition and to identify specific sequence(s) in the Glut4 molecule which target this protein into IRVs. We have also developed an in vitro budding assay for Glut4-vesicles. We propose to use this approach in order to reconstitute major trafficking steps of the Glut4 pathway in vitro and to study molecular mechanisms of these events. Membrane budding from endosomes usually requires a scaffold or a sorting receptor that gathers cargo proteins together and recruits protein coats to this complex. In the previous funding period, we have identified a novel component protein, sortilin, in the total pool of intracellular Glut4-vesicles. We suggest that this protein may play an important role in the organization of the insulin-sensitive glucose transporting machinery and, in particular, in formation of IRVs. Thus, we propose to study the biological role of sortilin in adipocytes using the antisense technique.